Dibromobimane

Guide (The following is our recommended protocol. This protocol is for guidance only and should be modified according to your specific needs).

1. Stock Solution Preparation
1.1 Solvent: 100% sterile-filtered dimethyl sulphoxide[1].
1.2 Concentration recommendation: 4 mM[1]; use the concentration appropriate for your experimental system.

2. Working Solution Preparation
2.1 Diluent: Sterile phosphate buffered saline, pre-warmed to 37°C.[1]
2.2 Working concentration: 40 μM.[1]
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.[1]

3. Staining Procedure
3.1 Sample type descriptions
3.1.1 For adherent mammalian cells (HEK 293T cells)[1]: No trypsinization needed; cells are cultured directly in 96-well microplates.
3.1.2 For reduced recombinant soluble tissue factor (sTF) protein (prepared via DTT reduction and desalting after blocking unpaired thiols with iodoacetamide)[2]: No additional sample preparation steps specified.
3.2 Incubation conditions
3.2.1 For adherent mammalian cells[1]: Incubate cells with 40 μM dibromobimane working solution at 37°C for 30 min.
3.2.2 For reduced recombinant sTF protein[2]: Incubate reduced sTF with 100 μM dibromobimane at room temperature for 30 min.
3.3 Washing steps
3.3.1 For adherent mammalian cells[1]: Drain media by inversion and gentle shaking, then blot plate on bench paper to remove residual media before adding dibromobimane working solution.
3.3.2 For reduced recombinant sTF protein[2]: No washing steps required.

4. Controls
4.1 Blank controls are included to subtract background fluorescence from experimental values.^[1]

5. Detection & Analysis
5.1 Instrument type: Fluorescent plate reader[1], fluorescence detection instrument[2].
5.2 Ex/Em wavelengths: Ex = 385-393 nm, Em = 477 nm.
5.3 Result analysis
5.3.1 For adherent mammalian cells[1]: Fluorescence intensity changes correlate with intracellular reduced glutathione levels; increased intensity indicates higher glutathione levels, while decreased intensity indicates lower glutathione levels. Final results are calculated as the ratio of dibromobimane fluorescence arbitrary units to Hoechst 33342 fluorescence arbitrary units for cell density normalization.
5.3.2 For reduced recombinant sTF protein[2]: Dibromobimane becomes fluorescent only after cross-linking closely spaced thiols, so fluorescent signal indicates successful labelling of reduced sTF's Cys186 and Cys209 thiols. Fluorescent signal corresponds to sTF protein bands resolved by SDS/PAGE.
5.3.3 Fluorescence emission is in the blue range (477 nm).

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.